HUMAN MUTATION Mutation in Brief #901 (2006) Online

MUTATION IN BRIEF

Mutation Analysis of COL1A1 and COL1A2 in Patients

Diagnosed With Osteogenesis Imperfecta Type I-IV
Rebecca Pollitt1, Robert McMahon1, Janice Nunn1, Robert Bamford1, Amal Afifi1, Nicholas Bishop2,
and Ann Dalton1
1
Sheffield Molecular Genetics Service, Sheffield Children’s NHS Trust, Sheffield, United Kingdom; 2Academic Unit of
Child Health, University of Sheffield, Sheffield, United Kingdom

*Correspondence to: Rebecca Pollitt, Sheffield Molecular Genetics Service, Sheffield Children’s NHS Trust, Western
Bank, Sheffield S10 2TH, United Kingdom; E-mail: rebecca.pollitt@sch.nhs.uk

Communicated by Mark H. Paalman

Osteogenesis Imperfecta (OI) is a heterogeneous group of inherited disorders characterized by

increased bone fragility, with clinical severity ranging from mild to lethal. To date, seven types
of OI have been described, based on clinical phenotype and histological findings. Most patients
with a clinical diagnosis of OI type I-IV have a mutation in the COL1A1 or COL1A2 genes
which encode the two alpha chains of type I collagen, the major component of the bone matrix.
Analysis of COL1A1 and COL1A2 in a cohort of 83 unrelated patients with OI type I-IV
identified a total of 62 mutations. Thirty-eight appear novel, 26 in COL1A1, and 12 in COL1A2,
and these are described here. The largest group consists of point mutations affecting glycine
residues in the triple helical domain of the two alpha chains, predicted to disrupt protein
folding and structure. This is in accordance with previously published data. A doublet GC
deletion, an unusual 398 base deletion predicted to completely remove exon 20 of COL1A2, and
a point mutation resulting in substitution of a conserved cysteine in the C-terminal propeptide
are described. In addition rare mutations at the cleavage sites of the C-propeptide and the N-
terminal signal peptide are described. © 2006 Wiley-Liss, Inc.

KEY WORDS: osteogenesis imperfecta; OI; COL1A1; COL1A2; type I collagen

INTRODUCTION
Osteogenesis imperfecta (OI; MIM# 166200; 166210; 166220) is a rare heterogeneous group of inherited disorders
characterized by low bone mass and increased bone fragility estimated to affect 1:15,000 to 1:20,000 individuals.
Extra-skeletal manifestations, which are variably associated with the disorder, are blue sclera, dentinogenesis
imperfecta, hyperlaxity of ligaments and skin, wormian bones and hearing loss. To date seven types of OI have been
described, based on clinical phenotype and histological findings, representing extreme variation in severity from one
individual to another. The most widely used classification is by Sillence (Sillence et al., 1979) which distinguishes
four main clinical phenotypes. Type I is the most common and is usually mild and non-deforming. Type II is the most
severe and is lethal in the perinatal period. Type III is severely deforming and fractures are often present at birth.
Type IV is moderately deforming, being between Type I and Type III in severity. Recently three further distinct
groups of moderate to severe phenotypes have been described, types V, VI and VII (Glorieux et al., 2000; Glorieux et
al., 2002; Ward et al., 2002).

Received 21 December 2005; accepted revised manuscript 16 March 2006.

© 2006 WILEY-LISS, INC.

DOI: 10.1002/humu.9430
2 Pollitt et al.

In more than 90% of patients with OI type I-IV the disorder is caused by an autosomal dominant mutation in one
of the two genes that encode the alpha chains of type I collagen, COL1A1 (MIM#120150; accession# for mRNA
Z74615.1) and COL1A2 (MIM# 120160; accession # for mRNA Z74616.1). Type I collagen is synthesized as a
soluble precursor, procollagen I, a heterotrimer consisting of two proα1 chains and one proα2 chain. Both proα1 and
proα2 chains consist of a triple helical domain of 1014 amino acids where glycine is invariantly in every third
position, forming a Gly-X-Y triplet repeat unit, which is flanked by globular carboxyl and amino terminal peptides.
Patients with a mild phenotype often have a strong family history of OI, although sporadic cases are reported. In
contrast severe phenotypes are usually de-novo and are most commonly associated with glycine substitutions in the
helical domain of the collagen molecule which are predicted to disrupt protein folding and structure. The phenotypic
effect of glycine mutations can be difficult to predict, as is the outcome of mutations in the conserved RNA splice
motif which have also been reported in OI of variable severities. Frameshift and nonsense mutations, which usually
result in reduced synthesis of normal collagen, result in a more predictable and milder phenotypic outcome. Rarely
mutations have been described in the C-propeptide domain. Here we describe thirty-eight novel mutations, including
mutations in the proα1 and proα2 C-propeptides and at the proα1 cleavage sites of the N-terminal signal peptide and
the C-propeptide.

MATERIALS AND METHODS

Patients
As part of ongoing service evaluation, a cohort of 83 unrelated patients referred to the Sheffield Molecular
Genetics Service for diagnostic screening was studied. Informed consent was obtained from each subject and/or legal
guardian. Patient’s parents and/or other family members were also studied in some instances in order to ascertain the
significance of some novel sequence changes, particularly where more than one potentially pathogenic change was
identified. None of the sequence changes reported here were detected in a normal control group (146 alleles).
The cohort consisted of 27 patients clinically diagnosed as OI type I, 15 type II, 13 type III and 16 type IV. Three
patients were classified borderline type I/IV. Nine patients had an unusual or unclear clinical phenotype.

Methods
Total genomic DNA was isolated from 2-5 mls peripheral blood using standard extraction methods. DNA
sequencing of PCR-amplified COL1A1 and COL1A2 gene fragments covering the entire coding region and
intron/exon boundaries was carried out using an ABI 3730 automated sequencer and Big Dye Terminator Sequencing
protocol (Applied Biosystems, Foster City, USA; http://www.appliedbiosystems.com). The nomenclature of
mutations identified was referenced to human mRNA for preproαI(I) collagen (GenBank accession number
Z74615.1) and human mRNA for preproα2(I) collagen (GenBank accession number Z74616.1), with the A of the
ATG translation initiation codon designated as +1, with the exception of an exon20del mutation which was referenced
to the genomic sequence for proα2(I) collagen (GenBank accession number AF004877).

RESULTS AND DISCUSION

A total of 62 mutations were identified in the patient cohort, 48 in COL1A1, and 14 in COL1A2. Thirty-eight of these
mutations appear to be novel (Dalgleish R, 1997; http://www.le.ac.uk/genetics/collagen) 26 in COL1A1 and 12 in
COL1A2, and these are described in Tables 1 and 2, respectively. The distribution of novel mutations relative to the
protein domains of proα1 and proα2 is summarized in Figure 1. The presenting phenotypes of patients with previously
reported mutations were in accordance with published data.
 Analysis of COL1A1 and COL1A2 in OI Patients 3

Table 1. Novel COL1A1 mutations in patients with OI type I-IV

Case OI Nucleotide change Predicted effect Exon
type #
Exonic substitution 443 II c.64G>C p.G22R 1
IV c.589G>C p.G197R 8
I c.959G>T p.G320V 15
IV c.1012G>T p.G338C 16
II c.1741G>A p.G581R 25
I/IV c.1939G>A p.G647S 29
II c.2201G>T p.G734V 32
I c.1414C>T p.R472X 21
922 I c.1664C>G p.P555R 24
IV c.3806G>A p.W1269X 49
943 I c.3657T>G, c.862G>A p.D1219E, p.E288K 49,13
231 II c.4237G>A p.D1413N 51
Insertion/deletion I c.386_387insC p.P129fsX39 5
IV c.386delC p.P129fsX135 5
I c.432delC p.P144fsX120 5
IV c.1379_1380insC p.P460fsX14 21
I c.1944delT p.P648fsX117 29
I c.3008delC p.P1003fsX104 42
IV c.3008delC p.P1003fsX104 42
299 III c.3580_3581delGC p.A1194fsX24 49
153 I c.3584delG p.G1195fsX43 49
I c.4054delC p.L1352fsX 51
816 I 17q21.33 to q23.1del COL1A1 del
Intronic substitution I c.471+1G>A Splicing variant 5
I c.589-2A>G Splicing variant 8
IV c.1876-2A>G Splicing variant 28
The position of the mutations refer to cDNA and protein sequences as they appear in GenBank sequence for human mRNA for
preproαI(I) collagen (GenBank accession number Z74615.1), with the A of the ATG translation initiation codon designated as
+1.
4 Pollitt et al.

Table 2. Novel mutations in COL1A2 in patients with OI type I-IV

Case OI Nucleotide change Predicted effect Exon
type #
Exonic Substitution IV c.604G>C p.G202R 13
I/IV c.739G>C p.G247R 16
II/III c.758G>A, c.4067G>A(1A1) p.G253D, p.R1356H (1A1) 16
IV c.767G>T p.G256V 16
I c.955G>A p.G319R 19
I c.2197G>T p.G733C 37
II c.2945G>A p.G982D 45
II c.3008G>A p.G1003D 46
III c.3260G>A p.G1087D 48
823 I c.3584G>A p.C1195Y 50
Insertion/deletion 104 II g.17390_17787del398 exon20del 20
Intronic substitution 189 II c.2944-2A>G, c.613C>G(1A1) Splice variant, p.P205A(1A1) 45
The position of the mutations refer to cDNA and protein sequences as they appear in GenBank sequence for human mRNA for
preproα2(I) collagen (GenBank accession number Z74616.1), with the A of translation initiation codon designated as +1, with
the exception of exon20del mutation which was referenced to the genomic sequence for proα2(I) collagen (GenBank accession
number AF004877).

The distribution of mutations in our patient cohort is similar to that reported in the literature, with the largest
group, 28/62, resulting in glycine substitution in the helical domain. Sixteen of these glycine substitutions appear to
be novel, seven in COL1A1 and nine in COL1A2, and are associated with phenotypes ranging from mild (type I) to
severe (type II).
Two regions in the alpha helix region of COL1A1, residues 691-823 and 910-964 (numbered from start of triple
helix) corresponding to major ligand binding regions, MLBR2 (682-830) and MLBR3 (920-1012), have been
identified as containing only lethal glycine mutations (Di Lullo et al., 2002). A patient who presented with a mild OI
phenotype was confirmed to have a sequence change resulting in a glycine substitution, p.G906S, located in
MLBR2. However it should be noted that this glycine forms one of only seven glycine doublets in COL1A1, three of
which lie within the helical domain, and occupies position X in the Gly-X-Y triplet repeat unit. OI phenotypes have
not previously been associated with glycine in the second or third position of the triplet repeat unit and the role of
this glycine in the structure of the triple helix is unknown. Although this change was not seen in our control group,
analysis of other family members is needed to clarify the significance of this unusual finding and samples have been
invited.
An unusual glycine mutation, p.G22R, at the cleavage recognition site of the N-terminal signal sequence of
COL1A1 (Tromp et al., 1988) resulting in a severe (type II) phenotype was identified in patient 443. There was no
evidence of the mutation in either parent, indicating that the mutation is de-novo. The N-terminal signal sequence is
required to transverse cellular membranes and its structure is important for recognition by the signal recognition
particle (SRP) and behavior in the membrane. The signal sequence is subsequently cleaved by signal peptidase.
Disruption of signal sequence cleavage as a consequence of this mutation may effect localization and/or processing
of the COL1A1 peptide.
Point mutations resulting in amino acid substitutions other than glycine were identified in phenotypes ranging
from mild (type I) to severe (typeII), and were located in the C-propeptide and helical domains. Low level paternal
mosaicism following two type II affected fetuses was demonstrated, using TaqI restriction digest, where the
 Analysis of COL1A1 and COL1A2 in OI Patients 5

mutation was confirmed as p.D1413N in the C-propeptide domain of COL1A1 (patient 231). This aspartic acid
residue is conserved in all fibrillar collagens and a similar mutation, p.D1441Y, has previously been reported to be
associated with a lethal variant of OI with features of dense bone disease (Pace et al., 2002).
.

p.D1219E
COL1A1 mutations
p.P555R (+ p.E288K) p.D1413N
p.G22R

N C

COL1A2 mutations p.C1195Y

Protein domains:

Signal sequence N and C telopeptides C-propeptide

N-propeptide containing
Triple helical domain
short helical domain

Mutations
Glycine mutations Non glycine exonic substitution
Splice variants Insertion/deletion mutations

Figure 1. Schematic representation of the distribution of novel mutations identified in COL1A1 (top) and
COL1A2 (bottom) relative to the functional domains of the proα1 and proα2 chains of type I collagen.
Mutations of note are indicated.

Of note is the putative mutation p.P555R in COL1A1 (patient 922), which was detected in five affected members
of a family where type I OI was segregating across 1st cousins. Proline and hydroxyproline are often in the second
and third position of the Gly-X-Y repeat unit and have been suggested to promote triple helix stability (Berg and
Prockop, 1973). Substitution of these residues for arginine has not previously been reported to result in an OI
phenotype and only two proline substitution have previously been reported p.P417A (Reis et al., 2005) and
p.P205A(Spotila et al.,1994) the former being associated with a mild OI phenotype without apparent metabolic bone
disease, the latter with a potential role in osteopenia. The association in this family between the disease and this
putative mutation would be expected by chance with a Baysian probability of less than 2%Substitution of a
conserved cysteine in the C-propeptide domain of COL1A2, p.C1195Y was detected in patient 823 diagnosed with
type I OI. The mutation was subsequently identified in two further affected family members and has been shown to
be segregating across 2nd cousins. This cysteine residue, one of seven in the C-propeptide domain of COL1A2,
participates in the complex pattern of disulphide links between neighboring proα1 and proα2 chains and is involved
in inter-chain covalent bond formation. Mutations that affect intra-chain disulphide bonds have been shown to result
in slowed chain association and excretion of overmodified procollagen molecules but lead to stable procollagen
trimers and a resultant mild phenotype (Pace et al. 2001). However, studies in type III procollagen suggest that, in
contrast, inter-chain disulphide bond formation is not required for chain association and triple-helix formation but
may play a role in maintaining procollagen stability (Bulleid et al., 1996). The effects of disruption to inter-chain
disulphide bonds caused by p.C1195Y and the exact mechanism by which the mild phenotype arises in this family
are unclear.
In patient 943 with a family history of OI type I a mutation at the proα1 C-propeptide cleavage site, p.D1219E
was identified with a second sequence change resulting in p.E288K. The Asp residue at position 1219 forms part of
6 Pollitt et al.

the Ala-Asp procollagen C-proteinase (pCP) cleavage site and is conserved in type I, II and III procollagens across
different species. This mutation is the first report of a mutation at the proα1 C-propeptide cleavage site and the
mechanism by which an OI phenotype arises will be dependent on the kinetics of pCP activity at the mutated
cleavage site and its effect on fibrillogenesis. Fibrils obtained from mice with null alleles for the procollagen c-
proteinase enhancer I gene (Pcolce-/-) which have markedly reduced efficiency of pCP cleavage showed
abnormalities in ultrastructure of collagen fibrils attributed to dysfunctional regulation at an early stage in
fibrillogenesis rather than to retention of C-propeptide in the collagen fibrils (Steiglitz et al., 2006) and this may
represent a model for fibrillogenesis in this patient. In contrast collagen produced in mice doubly null for Bmp1/Tll1,
and hence having no normal pCP activity, showed fibrils with retained c-propeptides. Replacement of the Asp
residue with Gly in proα2 has been shown to block pCP cleavage of the C-propeptide and result in molecules that
are incorrectly crossed linked (Lee et al., 1990). Whether this p.D1219E mutation is sufficient to block rather than
reduce the efficiency of pCP cleavage of the proα1 C-propeptide and if the resultant partially processed procollagen
molecules would then be incorporated into collagen fibrils is still to be resolved. Phenotypically the patient had nine
fractures by age ten and congenital cervical spondylosis. There is no evidence of joint hypermobility, dentinogenesis
imperfecta or hearing loss in the family. Analysis of five family members, three of whom are affected, has
confirmed that p.D1219E is segregating with the OI phenotype in this family. The p.E288K variant was identified in
the patient’s unaffected parent and is therefore probably a benign, family specific variant but a role as a phenotypic
modifier cannot be ruled out.
Patient 816 was reported to have a de-novo deletion of chromosome 17q21.33 to q23.1 demonstrated by FISH
analysis. He has micrognathia and cleft palate (Robin sequence), marfanoid features, blue sclerae and had a fracture
at birth with osteopenia on X-rays. Analysis of polymorphisms in the patient’s parents allowed for confirmation of a
deletion of COL1A1 on the paternal allele, giving rise to the mild OI phenotype. Isolated Robin sequence has been
suggested to result from dysregulation of SOX9 (17q24.3), a gene associated with Campomelic dysplasia (CD).
Separation of SOX9 from distant regulatory elements, which are known to lie up to 1Mb upstream, gives rise to
milder phenotypes with only some feature of CD (Hill-Harfe et al., 2005; Velagaleti et al., 2005) and this may
represent the mechanism for the non-OI related phenotypic features in this individual.
The doublet GC deletion c3580_3581delGC (patient 299), in contrast to the similar mutation c3584delG (patient
153), was an unexpected finding in a patient with a type III phenotype, suggesting that other genes may play a role
in phenotypic modification in this patient. An unusual 398bp deletion (patient 104) resulting in complete removal of
the 18 residues of exon 20 of COL1A2 but still leading to a triple helix that is predicted to remain in frame, gave rise
to a lethal phenotype.
Of note is the relatively high frequency of patients (5/83) in this cohort found to have more than one potentially
pathogenic or phenotype modifying sequence change. In the first, p.R882X with p.P978S, family studies have
shown that the premature termination codon at p.R882X is segregating with, and lies upstream of, p.P978S. Thus
p.P978S is unlikely to affect the resultant phenotype in this family. In the event of a recombination event the
resultant phenotype would be unpredictable, but possibly more severe. The p.D1219E C-propeptide cleavage site
mutation was identified with a second sequence change resulting in p.E288K as described above. One type II patient
(189) was shown to have an unusual splice site mutation c.2944-2A>G in COL1A2 in conjunction with p.P205A in
COL1A1, a variant reported to be associated with osteopenia. The interaction of this variant with the splice site
mutation and its role in the phenotype of this patient is unclear. Two further patients had mutations known to be
associated with OI phenotypes, in conjunction with second changes of unknown phenotypic consequence: p.Q957X
in COL1A1 with p.N1285H in COL1A2, and p.G253D in COL1A2 with p.A1356H in COL1A1.
The mutation detection rate for this cohort of patients, including those with unclear phenotypes, using this
method of sequencing the entire coding region and intron/exon boundaries of the type I collagen genes is 75%. For
patients with a clear phenotypic picture of OI the mutation detection rate is 84%. This is consistent with previously
published data.

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